Karyomegalic interstitial nephritis (KIN) is a chronic interstitial nephropathy characterized by tubulointerstitial nephritis and formation of enlarged nuclei in the kidneys and other tissues. We ...recently reported that recessive mutations in the gene encoding FANCD2/FANCI-associated nuclease 1 (FAN1) cause KIN in humans. FAN1 is a major component of the Fanconi anemia-related pathway of DNA damage response (DDR) signaling. To study the pathogenesis of KIN, we generated a Fan1 knockout mouse model, with abrogation of Fan1 expression confirmed by quantitative RT-PCR. Challenging Fan1
and wild-type mice with 20 mg/kg cisplatin caused AKI in both genotypes. In contrast, chronic injection of cisplatin at 2 mg/kg induced KIN that led to renal failure within 5 weeks in Fan1
mice but not in wild-type mice. Cell culture studies showed decreased survival and reduced colony formation of Fan1
mouse embryonic fibroblasts and bone marrow mesenchymal stem cells compared with wild-type counterparts in response to treatment with genotoxic agents, suggesting that FAN1 mutations cause chemosensitivity and bone marrow failure. Our data show that Fan1 is involved in the physiologic response of kidney tubular cells to DNA damage, which contributes to the pathogenesis of CKD. Moreover, Fan1
mice provide a new model with which to study the pathomechanisms of CKD.
Galloway‐Mowat syndrome (GAMOS) is a phenotypically heterogeneous disorder characterized by neurodevelopmental defects combined with renal‐glomerular disease, manifesting with proteinuria. To ...identify additional monogenic disease causes, we here performed whole exome sequencing (WES), linkage analysis, and homozygosity mapping in three affected siblings of an Indian family with GAMOS. Applying established criteria for variant filtering, we identify a novel homozygous splice site mutation in the gene WDR4 as the likely disease‐causing mutation in this family. In line with previous reports, we observe growth deficiency, microcephaly, developmental delay, and intellectual disability as phenotypic features resulting from WDR4 mutations. However, the newly identified allele additionally gives rise to proteinuria and nephrotic syndrome, a phenotype that was never reported in patients with WDR4 mutations. Our data thus expand the phenotypic spectrum of WDR4 mutations by demonstrating that, depending on the specific mutated allele, a renal phenotype may be present. This finding suggests that GAMOS may occupy a phenotypic spectrum with other microcephalic diseases. Furthermore, WDR4 is an additional example of a gene that encodes a tRNA modifying enzyme and gives rise to GAMOS, if mutated. Our findings thereby support the recent observation that, like neurons, podocytes of the renal glomerulus are particularly vulnerable to cellular defects resulting from altered tRNA modifications.
Myc, a member of the "Myc Network" of bHLH-ZIP transcription factors, supervises proliferation, metabolism, and translation. It also engages in crosstalk with the related "Mlx Network" to co-regulate ...overlapping genes and functions. We investigated the consequences of stepwise conditional inactivation of Myc and Mlx in primary and SV40 T-antigen-immortalized murine embryonic fibroblasts (MEFs).
-knockout (
KO) and
×
"double KO" (DKO)-but not
KO-primary MEFs showed rapid growth arrest and displayed features of accelerated aging and senescence. However, DKO MEFs soon resumed proliferating, indicating that durable growth arrest requires an intact Mlx network. All three KO MEF groups deregulated multiple genes and functions pertaining to aging, senescence, and DNA damage recognition/repair. Immortalized KO MEFs proliferated in Myc's absence while demonstrating variable degrees of widespread genomic instability and sensitivity to genotoxic agents. Finally, compared to primary
KO MEFs, DKO MEFs selectively downregulated numerous gene sets associated with the p53 and retinoblastoma (Rb) pathways and G
/M arrest. Thus, the reversal of primary
KO MEF growth arrest by either Mlx loss or SV40 T-antigen immortalization appears to involve inactivation of the p53 and/or Rb pathways.
Abstract Metastatic Ewing sarcoma has a very poor prognosis and therefore new investigations into the biologic drivers of metastatic progression are key to finding new therapeutic approaches. The ...tumor microenvironment is highly dynamic, leading to exposure of different regions of a growing solid tumor to changes in oxygen and nutrient availability. Tumor cells must adapt to such stress in order to survive and propagate. In the current study, we investigate how Ewing sarcoma cells respond to the stress of growth factor deprivation and hypoxia. Our findings reveal that serum deprivation leads to a reversible change in Ewing cell cytoskeletal phenotypes. Using an array of migration and invasion techniques, including gelatin matrix degradation invadopodia assays, we show that exposure of Ewing sarcoma cells to serum deprivation and hypoxia triggers enhanced migration, invadopodia formation, matrix degradation and invasion. Further, these functional changes are accompanied by and dependent on activation of Src kinase. Activation of Src, and the associated invasive cell phenotype, were blocked by exposing hypoxia and serum-deprived cells to the Src inhibitor dasatinib. These results indicate that Ewing sarcoma cells demonstrate significant plasticity in response to rapidly changing micro-environmental stresses that can result from rapid tumor growth and from necrosis-causing therapies. In response to these stresses, Ewing cells transition to a more migratory and invasive state and our data show that Src is an important mediator of this stress response. Our data support exploration of clinically available Src inhibitors as adjuvant agents for metastasis prevention in Ewing sarcoma.
Recessive mutations in the SDCCAG8 gene cause a nephronophthisis-related ciliopathy with Bardet-Biedl syndrome-like features in humans. Our previous characterization of the orthologous Sdccag8gt/gt ...mouse model recapitulated the retinal-renal disease phenotypes and identified impaired DNA damage response signaling as an underlying disease mechanism in the kidney. However, several other phenotypic and mechanistic features of Sdccag8gt/gt mice remained unexplored. Here we show that Sdccag8gt/gt mice exhibit developmental and structural abnormalities of the skeleton and limbs, suggesting impaired Hedgehog (Hh) signaling. Indeed, cell culture studies demonstrate the requirement of SDCCAG8 for ciliogenesis and Hh signaling. Using an affinity proteomics approach, we demonstrate that SDCCAG8 interacts with proteins of the centriolar satellites (OFD1, AZI1), of the endosomal sorting complex (RABEP2, ERC1), and with non-muscle myosin motor proteins (MYH9, MYH10, MYH14) at the centrosome. Furthermore, we show that RABEP2 localization at the centrosome is regulated by SDCCAG8. siRNA mediated RABEP2 knockdown in hTERT-RPE1 cells leads to defective ciliogenesis, indicating a critical role for RABEP2 in this process. Together, this study identifies several centrosome-associated proteins as novel SDCCAG8 interaction partners, and provides new insights into the function of SDCCAG8 at this structure.
Karyomegalic interstitial nephritis (KIN) is a genetic adult-onset chronic kidney disease (CKD) characterized by genomic instability and mitotic abnormalities in the tubular epithelial cells. KIN is ...caused by recessive mutations in the FAN1 DNA repair enzyme. However, the endogenous source of DNA damage in FAN1/KIN kidneys has not been identified. Here we show, using FAN1-deficient human renal tubular epithelial cells (hRTECs) and FAN1-null mice as a model of KIN, that FAN1 kidney pathophysiology is triggered by hypersensitivity to endogenous reactive oxygen species (ROS), which cause chronic oxidative and double-strand DNA damage in the kidney tubular epithelial cells, accompanied by an intrinsic failure to repair DNA damage. Furthermore, persistent oxidative stress in FAN1-deficient RTECs and FAN1 kidneys caused mitochondrial deficiencies in oxidative phosphorylation and fatty acid oxidation. The administration of subclinical, low-dose cisplatin increased oxidative stress and aggravated mitochondrial dysfunction in FAN1-deficient kidneys, thereby exacerbating KIN pathophysiology. In contrast, treatment of FAN1 mice with a mitochondria-targeted ROS scavenger, JP4-039, attenuated oxidative stress and accumulation of DNA damage, mitigated tubular injury, and preserved kidney function in cisplatin-treated FAN1-null mice, demonstrating that endogenous oxygen stress is an important source of DNA damage in FAN1-deficient kidneys and a driver of KIN pathogenesis. Our findings indicate that therapeutic modulation of kidney oxidative stress may be a promising avenue to mitigate FAN1/KIN kidney pathophysiology and disease progression in patients.
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